Application of ATG7 lactoacylation targeted metabolism reprogramming in tumor treatment

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a novel application for regulating and controlling de-lactation acylation of a K309 site of an autophagy-associated protein 7 (ATG7) based on a silent information regulatory factor 1 (Sirt1) activator and treating tumors through targeted metabolism reprogramming. The key effect of lactoacylation modification of the K309 site of ATG7 in tumor cell metabolism reprogramming is disclosed for the first time, and it is proved that a Sirt1 activator inhibits the Warburg effect through lactoacylation, enhances mitochondrial oxidative phosphorylation (OXPHOS) and triggers metabolic stress and tumor cell death. Cell experiments prove that the Sirt1 activator has a remarkable killing effect on tumor cells with high ATG7-K309 lactoacylation level, and a new strategy is provided for precise treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a method for regulating the delactylization of autophagy-related protein 7 (ATG7) based on a silent information regulator 1 (Sirt1) activator, and killing tumor cells by targeting metabolic reprogramming. Background Art

[0002] Even under aerobic conditions, tumor cells tend to use glycolysis rather than mitochondrial oxidative phosphorylation (OXPHOS) to obtain energy, and this phenomenon is called the Warburg effect. The Warburg effect provides necessary energy and nutrients for tumor cell proliferation. Tumor treatment targeting metabolic reprogramming is a new strategy that selectively intervenes in the energy metabolism pathway based on the characteristic that cancer cells rapidly proliferate through abnormal metabolism (such as the Warburg effect). Blocking the energy supply and biosynthesis of cancer cells while reducing damage to normal cells is a current research hotspot in precision tumor treatment.

[0003] Lactylation is a new type of post-translational protein modification mediated by lactic acid through non-enzymatic reactions or enzymatic reactions, but the role of lactylation in the Warburg effect has not been determined. Autophagy-related protein 7 (ATG7) is a core autophagy protein, and most of the research on the post-translational modification of ATG7 has focused on its autophagy-related functions. Currently, no study has revealed the regulation of the non-autophagy-dependent function of ATG7 by lactylation modification, nor is there a targeted regulation method.

[0004] Sirt1 is a NAD + -dependent histone deacetylase, which is involved in regulating energy metabolism, oxidative stress, and cell senescence. In recent years, it has been found that Sirt1 can affect the activity of target proteins through deacetylation or delactylization modification, but its mechanism of action in tumor metabolic reprogramming is not yet clear.

[0005] Currently, Sirt1 activators (such as resveratrol, SRT1720) are mainly used for anti-aging or the treatment of metabolic diseases, and their anti-tumor mechanisms mainly focus on deacetylation and have not involved lactylation modification regulation. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide an application of ATG7 lactylation targeting metabolic reprogramming in the treatment of tumors. It is first revealed that the lactylation modification at the K309 site of ATG7 plays a key role in tumor cell metabolic reprogramming. The Sirt1 activator can de-lactylize the K309 site of ATG7. De-lactylization leads to enhanced mitochondrial oxidative phosphorylation (OXPHOS) in tumor cells, while the glycolysis pathway (Warburg effect) is inhibited, triggering metabolic stress and cell death. Through cell experiments, it is demonstrated that the Sirt1 activator can inhibit the Warburg effect and thus kill tumor cells by de-lactylizing the K309 site of ATG7.

[0007] To achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0008] The first aspect of the present invention discloses the application of autophagy-related protein 7 with lactylation modification at lysine 309 as a target for treating tumors, and the target is a drug target or a target for regulating the tumor Warburg effect.

[0009] The second aspect of the present invention discloses the application of a de-lactylizing reagent for the K309 site of ATG7 in the preparation of a drug for treating tumors, and the de-lactylizing reagent includes a Sirt1 activator.

[0010] The third aspect of the present invention discloses the application of a Sirt1 activator in the preparation of a reagent for inhibiting the tumor Warburg effect.

[0011] The fourth aspect of the present invention discloses the application of a Sirt1 activator in the preparation of a de-lactylizing reagent for the K309 site of ATG7.

[0012] Furthermore, in the applications described above, the tumors include colon cancer and non-small cell lung cancer; the Sirt1 activators include resveratrol and SRT1720.

[0013] The fifth aspect of the present invention discloses a kit for detecting the lactylation level of ATG7K309 in tumor tissues, which is characterized by containing an antibody that specifically recognizes the lactylation modification of ATG7K309.

[0014] Furthermore, the kit is used for screening Sirt1 activator-sensitive tumors for treatment.

[0015] The sixth aspect of the present invention discloses a pharmaceutical composition for treating tumors with elevated ATG7-K309 lactylation level, and the pharmaceutical composition contains an effective amount of a Sirt1 activator and a pharmaceutically acceptable carrier.

[0016] Furthermore, the tumors include colon cancer and non-small cell lung cancer; the Sirt1 activators include resveratrol and SRT1720.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0018] The present invention firstly proposes that ATG7 has lactylation modification, and this modification occurs at the K309 site of the Atg7 protein. The delactylation targeting this site can inhibit the tumor Warburg effect and trigger the death of tumor cells.

[0019] Clinical application potential: By detecting the lactylation level of ATG7-K309 in the tumor tissues of patients, precise drug use can be achieved. Description of the Drawings

[0020] Figure 1 There is lactylation modification at the K309 site of the Atg7 protein. Among them, A shows that Atg7 can undergo lactylation modification; B shows that Atg7 can undergo lactylation modification; C shows that the lactylation modification of Atg7 occurs at the K309 site; D shows that the lactylation modification of Atg7 occurs at the K309 site.

[0021] Figure 2 It is about Sirt1 de-lactylation modifying the K309 site of the Atg7 protein. Among them, A shows that Sirt1 is the only de-lactylating enzyme in the Sirtuins family that can reduce the lactylation of Atg7; B shows that overexpression of Sirt1 can reduce the lactylation at the K309 site of the Atg7 protein; C shows that Sirt1 binds to Atg7 in the cell; D shows that Sirt1 binds to Atg7 in the cell; E shows that knocking out Sirt1 can increase the lactylation of Atg7; F shows that knocking down Sirt1 can increase the lactylation at the K309 site of the Atg7 protein; G shows that enhancing the activity of Sirt1 can reduce the lactylation level of Atg7; H shows that enhancing the activity of Sirt1 can reduce the lactylation level at the K309 site of the Atg7 protein; I shows that Sirt1 promotes the de-lactylation at the K309 site of the Atg7 protein; J shows that Sirt1 promotes the de-lactylation at the K309 site of the Atg7 protein.

[0022] Figure 3Deacylation of Atg7 at the K309 site under metabolic stress inhibits the Warburg effect in tumor cells. Among them, A shows that glucose deprivation can promote the deacylation of Atg7 protein; B shows that the deacylation of Atg7 protein at the K309 site caused by glucose deprivation is inhibited after knocking down Sirt1; C-E show the glycolysis rate, basal glycolysis, and compensatory glycolysis levels of H1299 cells stably expressing Flag-Atg7 and Flag-Atg7 K309R after culturing in a glucose-free medium for two hours; F-H show the oxygen consumption rate (OCR), basal respiration capacity, and ATP production rate levels of H1299 cells stably expressing Flag-Atg7 and Flag-Atg7 K309R after culturing in a glucose-free medium for two hours; I shows the CCK-8 assay results of H1299 cells stably expressing Flag-Atg7 and Flag-Atg7 K309R after glucose deprivation; J shows the colony formation assay results of H1299 cells stably expressing Flag-Atg7 and Flag-Atg7 K309R after glucose deprivation; K shows the CCK-8 assay results of H1299 cells stably expressing Flag-Atg7 and Flag-Atg7 K309R after treatment with SRT1720; L shows the colony formation assay results of H1299 cells stably expressing Flag-Atg7 and Flag-Atg7 K309R after treatment with SRT1720. Detailed implementation mode

[0023] The following takes specific examples to further describe the present invention in detail. However, this should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following examples, and all technologies implemented based on the content of the present invention belong to the scope of the present invention.

[0024] Unless otherwise specified, the reagents and materials used in the present invention are all commercially available.

[0025] I. Materials and methods.

[0026] 1. Cell culture.

[0027] The HEK293T cells, non-small cell lung cancer H1299 cell line, and colon cancer cell line HCT116 were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The HEK293T cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum, the H1299 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, and the HCT116 cells were cultured in McCoy's 5A medium containing 10% fetal bovine serum. The above cells were all cultured in a cell incubator at a constant temperature of 37 °C with 5% CO2.

[0028] 2. Plasmid construction, transfection, and lentivirus infection.

[0029] Flag-Atg7, Flag-Sirt1, Flag-Sirt2, Flag-Sirt3, Flag-Sirt4, Flag-Sirt5, Flag-Sirt6, Flag-Sirt7 were from our previous study. The Flag-Atg7 plasmid with the tetracycline-regulated Tet-on system was provided by GeneChem Co., Ltd. in Shanghai. We further generated the mutant Flag-Atg7 K309R plasmid with the Tet-on system by site-directed mutagenesis. The specific siRNA of Sirt1 was provided by Ribobio Co., Ltd. in Guangzhou. After confirmed by sequencing, they were transfected into cells using jetPrime transfection reagent and the corresponding jet buffer. For plasmid transfection, transfection reagents biobest or lipo3000 were used, and the cells were harvested at least 48 h later. After lentiviral infection, stable overexpressing and control cell lines were screened by G418 (400 μg / mL).

[0030] 3. Western blot and Co-IP

[0031] Cells were collected and lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate), and protease inhibitors were added to the lysis solution and phosphatase inhibitors were added as needed. After lysing for 30 min at 4°C, the mixture was centrifuged at 13,500 rpm for 20 min at 4°C, and the supernatant was collected. Subsequently, the protein was quantified by the Coomassie brilliant blue G-250 method or the BCA method, and protein samples were prepared according to the quantification results.

[0032] For co-immunoprecipitation, the procedures for cell lysis and preparation of the input sample were the same as those in the Western blot experiment. Subsequently, the primary antibody was added to the protein lysate and incubated at 4 °C for 1 h. Then, A / G magnetic beads (Santa Cruz) were added and mixed overnight at 4 °C. The next day, the magnetic beads were washed three times with PBS and then boiled in water with 2×loading buffer for 10 min, and the supernatant was collected. Subsequently, according to the molecular weight of the target protein, the protein samples were subjected to polyacrylamide gel electrophoresis in 8%, 10%, 12% or 15% SDS-PAGE gels and transferred to a PVDF membrane (Millipore, IPVH00010). After blocking with 5% bovine serum albumin (BSA) at room temperature for 1 h, it was washed three times with TBST and then incubated with the primary antibody overnight at 4 °C. Then, it was washed three times with TBST and incubated with the HRP-conjugated secondary antibody at room temperature for 2 h. Finally, after three washes, the enhanced chemiluminescence detection kit (Thermo Fisher Scientific, 32106) was used to detect the bands, and visualization was performed using the DNR Western blot detection system.

[0033] 4. Seahorse analysis.

[0034] To evaluate the effect of lactylation modification of Atg7 at the K309 site on its metabolism, we used the Seahorse Mito Stress detection kit to detect the oxygen consumption rate (OCR) and the Seahorse XF Glycolysis Rate assay kit to measure the glycolysis rate. These experiments were performed using the XF24 Extracellular Flux Analyzer from Seahorse Bioscience. H1299 cells stably overexpressing Atg7 or Atg7 K309R were seeded into XFe24-well microplates at a density of 1×10 5 cells per well and cultured overnight to adhere in a cell incubator at 37 °C with 5% CO2, during which 1 μg / mL doxycycline was added to induce the expression of Atg7. For Seahorse analysis, the sensor cartridge was hydrated overnight in a non-CO2 incubator at 37 °C using the Seahorse XF Calibrant. One hour after replacing the medium in the microplate with sugar-free medium, the medium in the cell culture plate was replaced with 500 μL of Seahorse XF RPMI or DMEM assay medium. Then, detection was performed after culturing in a non-CO2 incubator at 37 °C for 1 h. According to the manufacturer's protocol, the glycolysis rate and oxygen consumption rate were determined using the Seahorse XF Glycolysis Rate assay kit and the Seahorse Mito Stress detection kit, respectively.

[0035] 5. Cell proliferation assay and colony formation.

[0036] H1299 cells were seeded into 96-well plates at a density of 1×10 4 cells per well. After incubation in RPMI 1640 medium containing 10% fetal bovine serum for 24 h, the RPMI 1640 medium was replaced with glucose-free medium, or 10 µM, 12 µM, or 14 µM of SRT1720 was added. At the time of measurement, the glucose-free medium or RPMI 1640 medium was mixed with CCK8 (Cell Counting Kit-8) staining solution and then added to the cells in each well, and incubated at 37 °C for 2 h. The absorbance was measured at 450 nm using a microplate reader (TECAN, Switzerland), and the percentage of cell viability was calculated.

[0037] In the colony formation assay, H1299 cells were seeded into 6-well plates at a density of 2×10 4 cells per well and cultured in RPMI 1640 medium containing 10% fetal bovine serum. After 3 days, the RPMI 1640 medium was replaced with glucose-free medium, or 4 µM, 8 µM, or 10 µM of SRT1720 was added. After treatment, the cells were fixed with 4% paraformaldehyde at room temperature for 20 min, then stained with Coomassie Brilliant Blue (R250) at room temperature for 15 min, and imaged.

[0038] II. Experimental results.

[0039] 1. There is lactylation modification at the K309 site of Atg7 protein.

[0040] To investigate whether Atg7 is lactylated, we first performed immunoprecipitation experiments. Immunoprecipitation experiments were performed with Atg7 antibody in the 293T cell line, and whether Atg7 is lactylated was examined by a pan-lactylation antibody ( Figure 1 A). Immunoprecipitation experiments were performed with a pan-lactylation antibody in the 293T cell line, and whether Atg7 is lactylated was examined by Atg7 antibody ( Figure 1 B). The experimental results showed that Atg7 has lactylation modification.

[0041] According to the site suggested by the lactylation proteomics of Atg7, a lactylation-specific site antibody of Atg7, Atg7-K309 Lactyl Lysine, was produced, and we performed immunoprecipitation experiments. Immunoprecipitation experiments were performed with Atg7 antibody in the 293T cell line, and whether there is lactylation at the K309 site of Atg7 protein was examined by the specific lactylation site antibody ( Figure 1C). An immunoprecipitation experiment was performed using an antibody against the K309 site of the Atg7 protein in the 293T cell line, and the lactylation of the K309 site of the Atg7 protein was examined using the Atg7 antibody ( Figure 1 D). The experimental results showed that lactylation modification existed at the K309 site of the Atg7 protein.

[0042] 2. Sirt1 de-lactylates the K309 site of the Atg7 protein.

[0043] The Sirtus family is an important part of the acyltransferase family. Therefore, we overexpressed the Flag empty plasmid, Flag-Sirt1 plasmid, Flag-Sirt2 plasmid, Flag-Sirt3 plasmid, Flag-Sirt4 plasmid, Flag-Sirt5 plasmid, Flag-Sirt6 plasmid, and Flag-Sirt7 plasmid in 293T cells respectively. After 48 h, the cells were collected, and the proteins were enriched using the Pan Kla antibody and A / G beads. The lactylation changes of Atg7 were detected by western blot experiment. The experimental results showed that only overexpression of Sirt1 could reduce the lactylation of Atg7 ( Figure 2 A).

[0044] To further explore whether the de-lactylating effect of Sirt1 on Atg7 occurred at the K309 site of the Atg7 protein, we transfected the Flag empty plasmid and Flag-Sirt1 plasmid into HEK293 cells respectively. After 24 h, the cells were collected, the proteins were lysed, and the lactylation changes of the K309 site of the Atg7 protein were detected by western blot experiment. The experimental results showed that Sirt1 could reduce the lactylation of the K309 site of the Atg7 protein ( Figure 2 B).

[0045] To further confirm that Sirt1 is the de-lactylating enzyme of Atg7, we first detected the binding effect between Sirt1 and Atg7. 293T cells were collected and lysed, and the proteins were enriched using IgG antibody, Sirt1 antibody and A / G beads. The binding of Sirt1 and Atg7 was detected by western blot experiment ( Figure 2 C). 293T cells were collected and lysed, and the proteins were enriched using IgG antibody, Atg7 antibody and A / G beads. The binding of Atg7 and Sirt1 was detected by western blot experiment ( Figure 2 D). The results showed that there was an interaction between Sirt1 and Atg7.

[0046] To further clarify the de-lactylylation effect of Sirt1 on Atg7, we harvested HEK293T cells and HEK293T cells with Sirt1 knockout, and used Pan Kla antibody and A / G beads to enrich the proteins. The experimental results showed that the lactylation level of Atg7 in the HEK293T cell line with Sirt1 knockout was higher than that in the normal HEK293T cell line ( Figure 2 E).

[0047] To further clarify the de-lactylylation effect of Sirt1 on the K309 site of Atg7 protein, we transiently silenced Sirt1 using small interfering RNA. After 48 h, the cells were harvested and lysed for western blot analysis. An Atg-K309 site-specific lactylation antibody was used to detect the lactylation changes at the K309 site of Atg7 protein. The experimental results showed that the reduction of Sirt1 could increase the lactylation at the K309 site of Atg7 protein ( Figure 2 F).

[0048] To further determine the effect of the enzymatic activity of Sirt1 on the lactylation of Atg7, we administered SRT1720 to HEK293T cells and set up a control. After 24 h, the cells were harvested and lysed, and enriched with Pan Kla antibody and A / G beads. HEK293T cells were enriched with IgG antibody and A / G beads as a control, and western blot analysis was performed to detect the lactylation level of Atg7. The experimental results showed that activating Sirt1 could reduce the lactylation of Atg7 ( Figure 2 G).

[0049] To further determine the effect of the enzymatic activity of Sirt1 on the lactylation of the K309 site of Atg7 protein, we collected HEK293 cells after administering SRT1720 for 24 h, lysed the proteins, and performed western blot analysis to detect the lactylation changes at the K309 site of Atg7 protein. The experimental results showed that SRT1720 could reduce the lactylation at the K309 site of Atg7 protein ( Figure 2 H).

[0050] To further clarify the regulatory effect of Sirt1 on the lactylation of Atg7 at the K309 site, we transfected Flag-Sirt1 plasmid, myc-Atg7 plasmid / myc-Atg7 K309R plasmid in 293T cells. The medium was changed 4 - 6 h after transfection. Cells were harvested and lysed 48 h later. Proteins were enriched using Pan Kla antibody and A / G beads, and the lactylation expression levels of Atg7 and its mutant Atg7 K309R were detected by western blot. The experimental results showed that Sirt1 could reduce the lactylation of Atg7 but not that of the Atg-K309R mutant ( Figure 2 I).

[0051] To further clarify the regulatory effect of Sirt1 on the lactylation of Atg7 at the K309 site, we silenced Sirt1 using small interfering RNA. The myc-Atg7 plasmid / myc-Atg7 K309R plasmid was transfected 24 h later. Cells were harvested and lysed 24 h later, and co-immunoprecipitation experiments were performed. Proteins were enriched using Pan Kla antibody and A / G beads, and the lactylation changes of Atg7 and Atg7 K309R after knocking down Sirt1 were detected by western blot. The experimental results showed that knocking down Sirt1 could increase the lactylation of Atg7, while this increase in Atg7 lactylation was attenuated when the K309 site was mutated ( Figure 2 J).

[0052] 3. De-lactylation of Atg7 at the K309 site under metabolic stress inhibits the Warburg effect in tumor cells.

[0053] To investigate whether Atg7 lactylation modification is involved in the regulation of the tumor Warburg effect, we first detected the lactylation changes of Atg7 under glucose deprivation. HCT116 cells transfected with myc-Atg7 were treated with glucose deprivation for 0, 30, and 60 min, respectively. Cells were harvested and lysed, and proteins were enriched using Pan Kla antibody and A / G beads. The lactylation level of Atg7 was detected by western blot. The results showed that the lactylation of Atg7 gradually decreased with the prolongation of glucose deprivation time ( Figure 3 A).

[0054] To further clarify that glucose deprivation stimulates de-lactylylation of the K309 site of Atg7 protein by activating Sirt1, we transfected NC and Sirt1-siRNA into HCT116 cells. After 48 h, glucose deprivation treatment was given respectively, and the group without glucose deprivation treatment was used as a control. Cells were harvested and lysed, and the lactylation level of the K309 site of Atg7 protein was detected by western blot. The experimental results showed that glucose deprivation could lead to de-lactylylation of the K309 site of Atg7 protein, and this effect was inhibited after Sirt1 knockdown ( Figure 3 B).

[0055] To further explore the effect of Atg7 de-lactylylation on the Warburg effect of tumor cells under metabolic stress, we cultured H1299 cells stably expressing Flag-Atg7 and Flag-Atg7 K309R in a glucose-free medium for two hours, and then used a glycolysis rate kit to detect the glycolysis rate, basal glycolysis and compensatory glycolysis levels. The experimental results showed that when the K309 site of Atg7 was mutated to R, the inhibition levels of glycolysis rate, basal glycolysis and compensatory glycolysis of tumor cells in the face of metabolic stress decreased ( Figure 3 C-E).

[0056] To further detect the effect of Atg7 de-lactylylation on aerobic respiration of tumor cells under metabolic stress, we cultured H1299 cells stably expressing Flag-Atg7 and Flag-Atg7 K309R in a glucose-free medium for two hours, and then used a mitochondrial stress kit to detect the oxygen consumption rate (OCR), basal respiratory capacity and ATP production rate levels. The experimental results showed that when the K309 site of Atg7 was mutated to R, the oxygen consumption rate (OCR), basal respiratory capacity and ATP production rate levels of tumor cells in the face of metabolic stress decreased ( Figure 3 F-H).

[0057] To further explore the effect of Atg7 de-lactylylation on the survival of tumor cells under metabolic stress, we stably overexpressed Flag-Atg7 and Flag-Atg7 K309R in H1299 cells, and gave glucose deprivation stimulation for 18 h, 24 h, 30 h, and 36 h. The cell counting kit (CCK-8) method was used to detect the cell survival rate. The experimental results showed that under different glucose deprivation time gradients, the degree of inhibition of the Warburg effect of Atg7-K309R cells was lower than that of Atg7-WT cells, and glucose was depleted faster, resulting in cell death ( Figure 3 I).

[0058] To further clarify the effect of Atg7 delactylation on the survival of tumor cells under metabolic stress, we conducted a colony formation assay. We stably overexpressed Flag-Atg7 and Flag-Atg7 K309R in H1299 cells, and stimulated them with deglucosylation for 18 h, 24 h, and 30 h. After R250 staining, we observed whether there were differences in cell survival. The experimental results showed that under different deglucosylation time gradients, the degree of inhibition of the Warburg effect in Atg7-K309R cells was lower than that in Atg7-WT cells, and glucose was depleted faster, leading to cell death ( Figure 3 J).

[0059] To further explore the effect of Atg7 delactylation on the survival of tumor cells, we stably overexpressed Flag-Atg7 and Flag-Atg7 K309R in H1299 cells, treated them with different concentration gradients of 10 µM, 12 µM, and 14 µM of SRT1720, and used the untreated group as a control. The cell counting kit (CCK-8) method was used to detect cell viability. The experimental results showed that Atg7-K309R cells could not be delactylated by Sirt1, and SRT1720 had a worse killing effect on Atg7-K309R cells ( Figure 3 K).

[0060] To further clarify the effect of Atg7 delactylation on the survival of tumor cells under metabolic stress, we conducted a colony formation assay. H1299 cells were stably overexpressed with Flag-Atg7 and Flag-Atg7 K309R, treated with different concentration gradients of 10 µM, 12 µM, and 14 µM of SRT1720, and the untreated group was used as a control. After 24 h, R-250 staining was used to observe whether there were differences in cell survival. The experimental results showed that Atg7-K309R cells could not be delactylated by Sirt1, and SRT1720 had a worse killing effect on Atg7-K309R cells ( Figure 3 L).

[0061] The above are only the preferred embodiments of the present invention and are not used to limit the patent scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of autophagy-related protein 7 with lactylated lysine at position 309 as a target for treating tumors, wherein the target is a drug target or a target for regulating the Warburg effect of tumors.

2. Use of a delactylating agent for ATG7 K309 site in the preparation of a drug for treating tumors, wherein the delactylating agent comprises a Sirt1 activator.

3. Application of Sirt1 activators in the preparation of reagents for inhibiting the Warburg effect of tumors.

4. Application of Sirt1 activator in the preparation of de-lactylating reagent for ATG7 K309 site.

5. The use according to any one of claims 1 to 4, characterized in that: The tumors include colon cancer and non-small cell lung cancer; the Sirt1 activators include resveratrol and SRT1720.

6. A kit for detecting the lactylation level of ATG7K309 in tumor tissue, characterized in that: Contains an antibody that specifically recognizes the lactylated modification of ATG7K309.

7. The kit according to claim 6, characterized in that: The kit is used for screening Sirt1 activator for treating sensitive tumors.

8. A pharmaceutical composition for treating a tumor with elevated ATG7-K309 lactylation level, the pharmaceutical composition comprising an effective amount of a Sirt1 activator and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that The tumors include colon cancer and non-small cell lung cancer; the Sirt1 activators include resveratrol and SRT1720.

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